Optimizing the efficacy of epitope-directed DNA vaccination

An increasing number of clinical trials has been initiated to test the potential of prophylactic or curative vaccination with tumor Ag-encoding DNA vaccines. However, in the past years it has become apparent that for many Ags and in particular for tumor Ags the intracellular processing and presentation are suboptimal. To improve epitope-directed DNA vaccines we have developed a murine model system in which epitope-specific, DNA vaccine-induced T cell immunity can be followed by MHC tetramer technology directly ex vivo. We have used this well-defined model to dissect the parameters that are crucial for the induction of strong cytotoxic T cell immunity using two independent model Ags. These experiments have led to a set of five guidelines for the design of epitope-directed DNA vaccines, indicating that carboxyl-terminal fusion of the epitope to a carrier protein of foreign origin is the most favorable strategy. DNA vaccines that are based on these guidelines induce high-magnitude CD8(+) T cell responses in >95% of vaccinated animals. Moreover, T cell immunity induced by this type of optimized DNA vaccine provides long-term protection against otherwise lethal tumor challenges.     

The Pneumocystis carinii drug target S-adenosyl-L-methionine:sterol C-24 methyl transferase has a unique substrate preference

Pneumocystis is an opportunistic pathogen that can cause pneumonitis in immunodeficient people such as AIDS patients. Pneumocystis remains difficult to study in the absence of culture methods for luxuriant growth. Recombinant protein technology now makes it possible to avoid some major obstacles. The P. carinii expressed sequence tag (EST) database contains 11 entries of a sequence encoding a protein homologous to S-adenosyl-L-methionine (SAM):C-24 sterol methyl transferase (SMT), suggesting high activity of this enzyme in the organism. We sequenced the erg6 cDNA, identified the putative peptide motifs for the sterol and SAM binding sites in the deduced amino acid sequence and expressed the protein in Escherichia coli. Unlike SAM:SMT from other organisms, the P. carinii enzyme had higher affinities for lanosterol and 24-methylenelanosterol than for zymosterol, the preferred substrate in other fungi. Cycloartenol was not a productive substrate. With lanosterol and 24-methylenelanosterol as substrates, the major reaction products were 24-methylenelanosterol and pneumocysterol respectively. Thus, the P. carinii SAM:SMT catalysed the transfer of both the first and the second methyl groups to the sterol C-24 position, and the substrate preference was found to be a unique property of the P. carinii SAM:SMT. These observations, together with the absence of SAM:SMT among mammals, further support the identification of sterol C-24 alkylation reactions as excellent targets for the development of drugs specifically directed against this pathogen.     

Improved Estimation of Cardiac Function Parameters Using a Combination of Independent Automated Segmentation Results in Cardiovascular Magnetic Resonance Imaging

This work aimed at combining different segmentation approaches to produce a robust and accurate segmentation result. Three to five segmentation results of the left ventricle were combined using the STAPLE algorithm and the reliability of the resulting segmentation was evaluated in comparison with the result of each individual segmentation method. This comparison was performed using a supervised approach based on a reference method. Then, we used an unsupervised statistical evaluation, the extended Regression Without Truth (eRWT) that ranks different methods according to their accuracy in estimating a specific biomarker in a population. The segmentation accuracy was evaluated by estimating six cardiac function parameters resulting from the left ventricle contour delineation using a public cardiac cine MRI database. Eight different segmentation methods, including three expert delineations and five automated methods, were considered, and sixteen combinations of the automated methods using STAPLE were investigated. The supervised and unsupervised evaluations demonstrated that in most cases, STAPLE results provided better estimates than individual automated segmentation methods. Overall, combining different automated segmentation methods improved the reliability of the segmentation result compared to that obtained using an individual method and could achieve the accuracy of an expert.     

Leishmania infantum Modulates Host Macrophage Mitochondrial Metabolism by Hijacking the SIRT1-AMPK Axis

Metabolic manipulation of host cells by intracellular pathogens is currently recognized to play an important role in the pathology of infection. Nevertheless, little information is available regarding mitochondrial energy metabolism in Leishmania infected macrophages. Here, we demonstrate that during L. infantum infection, macrophages switch from an early glycolytic metabolism to an oxidative phosphorylation, and this metabolic deviation requires SIRT1 and LKB1/AMPK. SIRT1 or LBK1 deficient macrophages infected with L. infantum failed to activate AMPK and up-regulate its targets such as Slc2a4 and Ppargc1a, which are essential for parasite growth. As a result, impairment of metabolic switch caused by SIRT1 or AMPK deficiency reduces parasite load in vitro and in vivo. Overall, our work demonstrates the importance of SIRT1 and AMPK energetic sensors for parasite intracellular survival and proliferation, highlighting the modulation of these proteins as potential therapeutic targets for the treatment of leishmaniasis.     

Relation between Red Cell Distribution Width and Fibroblast Growth Factor 23 Cleaving in Patients with Chronic Kidney Disease and Heart Failure

Objective

In chronic kidney disease (CKD), both anemia and deregulated phosphate metabolism are common and predictive of adverse outcome. Previous studies suggest that iron status influences phosphate metabolism by modulating proteolytic cleavage of FGF23 into C-terminal fragments. Red cell distribution width (RDW) was recently identified as a strong prognostic determinant for cardiovascular morbidity and mortality, independently of iron status. We assessed whether RDW is associated with FGF23 cleaving in CKD patients with heart failure.

Materials and Methods

The associations between RDW and either intact FGF23 (iFGF23), C-terminal FGF23 (cFGF23, reflecting iFGF23 and C-terminal fragments together) and the iFGF23/cFGF23 ratio were analyzed in 52 patients with CKD (eGFR 34,9 ± 13.9 ml/min/1.73m²) and chronic heart failure (CHF). Associations between RDW and FGF23 forms were studied by linear regression analysis adjusted for parameters of renal function, iron metabolism, phosphate metabolism and inflammation.

Results

Median cFGF23 levels were 197.5 [110–408.5] RU/ml, median iFGF23 levels were 107.3 [65.1–162.2] pg/ml and median FGF23 ratio was 0.80 [0.37–0.86]. Mean RDW was 14.1 ± 1.2%. cFGF23 and RDW were associated (β = 1.63x10^-3, P <0.001), whereas iFGF23 and RDW were not (β = -1.38x10^-3, P = 0.336). The iFGF23/cFGF23 ratio was inversely associated with RDW. The difference between cFGF23 and iFGF23 (cFGF23- iFGF23) was positively associated with RDW (β = 1.74x10^-3, P< 0.001). The association between cFGF23 and RDW persisted upon multivariable linear regression analysis, adjusted for parameters of renal function, phosphate metabolism, iron metabolism and inflammation (β = 0.97x10^-3, P = 0.047).

Conclusion

RDW is associated with cFGF23 but not with iFGF23 levels in patients with CKD and CHF. This suggests a connection between RDW and FGF23 catabolism, independent of iron status and inflammation. Future studies are needed to unravel underlying mechanisms and whether these pertain to the link between RDW and outcome.     

Oxidation of hydrogen sulfide remains a priority in mammalian cells and causes reverse electron transfer in colonocytes

Sulfide (H₂S) is an inhibitor of mitochondrial cytochrome oxidase comparable to cyanide. In this study, poisoning of cells was observed with sulfide concentrations above 20 µM. Sulfide oxidation has been shown to take place in organisms/cells naturally exposed to sulfide. Sulfide is released as a result of metabolism of sulfur containing amino acids. Although in mammals sulfide exposure is not thought to be quantitatively important outside the colonic mucosa, our study shows that a majority of mammalian cells, by means of the mitochondrial sulfide quinone reductase (SQR), avidly consume sulfide as a fuel. The SQR activity was found in mitochondria isolated from mouse kidneys, liver, and heart. We demonstrate the precedence of the SQR over the mitochondrial complex I. This explains why the oxidation of the mineral substrate sulfide takes precedence over the oxidation of other (carbon-based) mitochondrial substrates. Consequently, if sulfide delivery rate remains lower than the SQR activity, cells maintain a non-toxic sulfide concentration (< 1 µM) in their external environment. In the colonocyte cell line HT-29, sulfide oxidation provided the first example of reverse electron transfer in living cells, such a transfer increasing sulfide tolerance. However, SQR activity was not detected in brain mitochondria and neuroblastoma cells. Consequently, the neural tissue would be more sensitive to sulfide poisoning. Our data disclose new constraints concerning the emerging signaling role of sulfide.     

Testing ecological and behavioral correlates of nest predation

Variation in nest predation rates among bird species are assumed to reflect differences in risk that are specific to particular nest sites. Theoretical and empirical studies suggest that parental care behaviors can evolve in response to nest predation risk and thereby differ among ecological conditions that vary in inherent risk. However, parental care also can influence predation risk. Separating the effects of nest predation risk inherent to a nest site from the risk imposed by parental strategies is needed to understand the evolution of parental care. Here we identify correlations between risks inherent to nest sites, and risk associated with parental care behaviors, and use an artificial nest experiment to assess site-specific differences in nest predation risk across nesting guilds and between habitats that differed in nest predator abundance. We found a strong correlation between parental care behaviors and inherent differences in nest predation risk, but despite the absence of parental care at artificial nests, patterns of nest predation risk were similar for real and artificial nests both across nesting guilds and between predator treatments. Thus, we show for the first time that inherent risk of nest predation varies with nesting guild and predator abundance independent of parental care.     

Structural and functional characterization of enantiomeric glutamic acid derivatives as potential transition state analogue inhibitors of MurD ligase

Mur ligases play an essential role in the intracellular biosynthesis of bacterial peptidoglycan, the main component of the bacterial cell wall, and represent attractive targets for the design of novel antibacterials. UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase (MurD) catalyses the addition of D-glutamic acid to the cytoplasmic intermediate UDP-N-acetylmuramoyl-L-alanine (UMA) and is the second in the series of Mur ligases. MurD ligase is highly stereospecific for its substrate, D-glutamic acid (D-Glu). Here, we report the high resolution crystal structures of MurD in complexes with two novel inhibitors designed to mimic the transition state of the reaction, which contain either the D-Glu or the L-Glu moiety. The binding modes of N-sulfonyl-D-Glu and N-sulfonyl-L-Glu derivatives were also characterised kinetically. The results of this study represent an excellent starting point for further development of novel inhibitors of this enzyme.     

Chronic lithium administration attenuates up-regulated brain arachidonic acid metabolism in a rat model of neuroinflammation

Neuroinflammation, caused by a 6-day intracerebroventricular infusion of lipopolysaccharide (LPS) in rats, is associated with the up-regulation of brain arachidonic acid (AA) metabolism markers. Because chronic LiCl down-regulates markers of brain AA metabolism, we hypothesized that it would attenuate increments of these markers in LPS-infused rats. Incorporation coefficients k* of AA from plasma into brain, and other brain AA metabolic markers, were measured in rats that had been fed a LiCl or control diet for 6 weeks, and subjected in the last 6 days on the diet to intracerebroventricular infusion of artificial CSF or of LPS. In rats on the control diet, LPS compared with CSF infusion increased k* significantly in 28 regions, whereas the LiCl diet prevented k* increments in 18 of these regions. LiCl in CSF infused rats increased k* in 14 regions, largely belonging to auditory and visual systems. Brain cytoplasmic phospholipase A(2) activity, and prostaglandin E(2) and thromboxane B(2) concentrations, were increased significantly by LPS infusion in rats fed the control but not the LiCl diet. Chronic LiCl administration attenuates LPS-induced up-regulation of a number of brain AA metabolism markers. To the extent that this up-regulation has neuropathological consequences, lithium might be considered for treating human brain diseases accompanied by neuroinflammation.